Superparamagnetic particles, process for their manufacture and usage

ABSTRACT

Superparamagnetic particles consist of superparamagnetic one-domain particles and aggregates of superparamagnetic one-domain particles to whose surfaces are bound organic substances optionally having further binding sites for coupling to tissue-specific binding substances, diagnostic or pharmacologically active substances. The superparamagnetic particles consist of a mixture of small superparamagnetic one-domain particles with a particle size from 3 to 50 nanometers and stable, degradable aggregates of small superparamagnetic one-domain particles with a particle size from 10 to 1000 nanometers. They are made of iron hydroxide, iron oxide hydrate, iron oxides, iron mixed oxides or iron to the surface of which are bound mono- and/or polyhydroxylic group-containing aromatic substances, polyglycerines, amino-acid-containing substances, silicate group-containing substances among the orthosilicic acids and their condensation products and phosphate group-containing substances among the ortho- or metaphosphoriic acids and their condensation products. These substances may have further binding sites. These new particles may be used to destroy tumors, increase immunity, in magnetic drug targeting, for cell fusion, gene transfers, as contrasting agents, for in vitro diagnosis, as magnetic ion exchangers and magnetic adsorbents, if required by exposure to magnetic fields.

The present invention relates to superparamagnetic particles consistingof superparamagnetic one-domain particles and aggregates ofsuperparamagnetic one-domain particles of iron oxides, mixed iron oxidesor iron to whose surfaces are bound organic substances which optionallymay have further binding sites for coupling tissue-specific bindingsubstances, diagnostic substances or pharmacologically activesubstances.

Patent EP-B-0284549 describes superparamagnetic one-domain particles ofiron oxide, mixed iron oxide or iron with a particle size ranging from 3to 20 nanometers, to whose surfaces are chemically bound organicsubstances of the group of phosphate, diphosphate, polyphosphate,thiophosphate, phosphonate or thiophosphonate group-containingpolyalkylene glycols, phosphate group-containing nucleotides, theiroligomers or their polymers as well as phosphate group-containingcarbohydrates, which may have further binding sites.

Patent DE-A-4309333 describes stable, degradable aggregates with aparticle size ranging from between 10 and 1000 nanometers with definedbehaviour in a magnetic field whereby the aggregates consist of severalsmall superparamagnetic one-domain particles of iron oxide, mixed ironoxide or iron with a particle size ranging from 3 to 20 nanometers towhose surfaces are chemically bound substances of the group ofphosphate, diphosphate, polyphosphate, thiophosphate, phosphonate orthiophosphonate group-containing polyalkylene glycols, carbohydrates orof the phosphate group-containing nucleotides, their oligomers or theirpolymers.

The object of the invention is to enlarge the range of substances whichcan be bound to the surfaces of the one-domain particles, in order tooptimally adapt the physicochemical and physiological properties of themagnetic particles being formed to the respective fields of applicationwhereby these substances should be stable and easy to manufacture.

A surprising finding was that mono- and polyhydroxylic group-containingaromatic substances, polyglycerin and their phosphate, diphosphate,polyphosphate, thiophosphate, phosphonate, thiophosphonate, carboxylate,sulphate, mercapto or silantriol group-containing derivatives, aminoacid containing oligopeptides, polypeptides, proteins, proteids as wellas their derivatives and denaturation products, mercapto and aminogroup-containing substances such as biotin, mercaptopurine, -cytosin,-guanine, -uracil, -thymine, -hypoxanthine, as well as theirmercaptonucleosides and mercapto-desoxynucleosides, ortho-silicic acidand their condensation products with bivalent and polyvalent inorganicions and/or organic acids and bases and ortho- or metaphosphoric acidand their condensation products enter stable bindings with the surfaceof the superparamagnetic particles leading to stable magnetic liquidsand aggregation-stable, superparamagnetic aggregates.

According to the invention, stabilisation of the magnetic particlestakes place by binding mono- and polyhydroxylic group-containingaromatic substances such as for example benzenoids, coumarins, lignans,terphenyls, flavonoids, tannins, xanthenes, benzophenones, naphthalenes,naphthoquinones, anthraquinones, anthracyclines, polycyclic condensatedaromatic compounds and their phosphate, diphosphate, polyphosphate,thiophosphate, phosphonate, thiophosphonate, carboxylate, sulphate,mercapto or silantriol group-containing derivatives, of polyhydroxylicgroup-containing substances, selected from among polyglycerins and theirphosphate, diphosphate, polyphosphate, thiophosphate, phosphonate,thiophosphonate, carboxylate, sulphate, mercapto or silantriolgroup-containing derivatives, of amino acid containing substances suchas for example oligopeptides, polypeptides, proteins, proteids as wellas their derivatives and denaturation products, of mercapto- and aminogroup-containing substances such as for example biotin, mercaptopurine,-cytosin, -guanine, -uracil, -thymine, -hypoxanthine, as well as theirmercapto-nucleosides and mercapto-desoxynucleosides, of silicategroup-containing substances of ortho-silicic acid and their condensationproducts with bivalent and polyvalent inorganic ions an/or organic acidsand bases, of phosphate group containing substances of ortho- ormetaphosphoric acid and their condensation products, on the surface ofthe magnetic particles.

Apart from these stabiliser substances, it is possible to bindadditional organic substances to the surface of the magnetic particlesin order to better adjust the properties of the magnetic particles tothe desired requirements. This includes organic substances of the groupof phosphate, diphosphate, carboxylate, polyphosphate, thiophosphate,phosphonate, thiophosphonate, sulphate, sulphonate, mercapto,carboxylate, silantriol group-containing polyalkylene glycols, andcarbohydrates, of the phosphate group-containing nucleotides, theiroligomers or their polymers, of the alkyl, aryl and/oralkyl-aryl-polyethylene glycol phosphates or phosphonates, of the groupof nitrogenous polysaccharides, selected from among mucopolysaccharides,glycoproteids, chitins, as well as their derivatives and denaturationproducts.

The following are examples of stabiliser substances for mono- and/orpolyhydroxyl group-containing aromatic substances: caffeic acid, gallicacid, hexahydroxydiphenic acid, ellagic acid, chebulic acid, and theirderivatives and denaturation products with carbohydrates and phenolcarbonic acids, aesculin, rutin, aescin, troxerutin, hesperidin, aloin,kaempferol, quercetin, gallotannin, ellagitannin, ruberythrinic acid,carminic acid, natural and synthetic dyes such as anthraquinone orphthalocyanin dyes, daunorubicin, ansamycin as well as their phosphate,diphosphate, polyphosphate, thiophosphate, phosphonate, thiophosphonate,carboxylate, sulphate, mercapto or silantriol group-containingderivatives.

The following are examples of stabiliser substances for substancescontaining amino acid of the groups of oligopeptides, polypeptides,proteins, proteids as well as their denaturation products: protamines,glutelins, albumins, globulins, gelatine, casein-hydrolysates.

Examples of stabiliser substances for substances containing mercapto andamino groups are: biotin, cysteine, methionine, glutathione,mercaptopurine, -cytosin, -guanine, -uracil, -thymine, -hypoxanthine, aswell as their mercaptonucleosides and mercapto-desoxynucleosides.

By way of example, stabiliser substances for silicate group-containingcondensation products of ortho-silicic acid with bi- and polyvalentinorganic ions are the condensation products with the elements Al, Au,Bi, Cr, J, Mo, P, Pt, Se, Tc, Ti, Y, Zr and rare precious metals.

By way of example, stabiliser substances for silicate group-containingcondensation products of ortho-silicic acid with organic acids and basesare their condensation products with phytic acid, alginic acid, gallicacid.

By way of example, stabiliser substances for phosphate group-containingcondensation products of ortho- or metaphosphoric acid arepyrophosphoric acid, polyphosphoric acids, cyclophospates and heterocondensation products and water insoluble salt compounds with inorganicions, such as Ag, Au, Bi, Mo, Pt, Tc, Y, Zr, and basic group-containingorganic compounds such as spermine, spermidine, polyethylene imine,oxygelatine.

The stabiliser substances may be manufactured according to the currentstate of technology, or purchased.

According to the invention, to the stabilisation molecules--to whosesurfaces are bound hydroxyl group-containing aromatic substances,polyhydroxyl group-containing polyglycerins, amino acid containingsubstances, silicate group-containing substances and phosphategroup-containing substances at the surface of the super paramagneticparticles--the following can be bound: tissue-specific bindingsubstances such as for example antigens, antibodies, ribonucleic acids,desoxyribonucleic acids, ribonucleic acid sequences, desoxyribonucleicacid sequences, haptens, avidin, streptavidin, protein A, protein G,endotoxin-binding proteins, lectin, selectin, pharmacologically activesubstances such as for example anti tumour proteins, enzymes, antitumour enzymes, antibiotics, plant alkaloids, alkylation reagents,antimetabolites, hormones and hormone antagonists, interleukins,interferons, growth factors, tumour necrosis factors, endotoxins,lymphotoxins, urokinase, streptokinase, plasminogen-streptokinaseactivator complex, tissue plasminogen activators, desmodus plasminogenactivators, macrophage activating bodies, antisera, protease inhibitors,substances containing radioactive isotopes, tensides, cardiovascularpharmaceutic products, chemotherapeutic products, gastrointestinalpharmaceutic products, neuropharmaceutic products, pharmacologicallyactive cells such as for example organelles, viruses, microbes, algae,fungi, in particular erythrocytes, thrombocytes, granulocytes,monocytes, lymphocytes, Langerhans islands, pharmacologically activecomplexing agents, such as for example polycarbonic acids,polyaminocarboxylic acids, porphyrins, catecholamines, cell-fusioningsubstances such as for example polyethylene glycols, alkyl polyethyleneglycols, alkyl aryl polyethylene glycols, water-miscible polypropyleneglycols (PPG)_(m) or water-miscible substance copolymerides ofpolyethylene glycol (PEG) and polypropylene glycol (PPG), selected fromamong the substance copolymerides (PEG)_(n) -(PEG)_(m), (PEG)_(n)-(PPG)_(m) -(PEG)_(n), (PPG)_(m) -(PEG)_(n) -(PPG)_(m) whereby n and mare positive whole numbers, selected for PEG in the range of 4 to 1000,for PPG in the range of 3 to 12 and for PEG-PPG substance copolymeridesin the range 3 to 140, and/or substances acting as gene-transfer media,such as for example polyethylene glycols and their derivatives andpolyalkylene imines, such as for example pentaethylene hexamine,polyethylene imine, spermine, spermidine.

The superparamagnetic particles according to the invention, which werestabilised with hydroxylic group-containing aromatic stabilisersubstances, adsorb compounds with a relatively high content of aminoacid and aromatic compounds, so that in some applications a purelyadsorptive binding of tissue-specific binding substances,pharmacologically active substances and pharmacologically active cellsis sufficient to be able to apply them for magnetic drug targeting or asa contrast medium.

The superparamagnetic particles according to the invention, which werestabilised with polyglycerins and their derivatives, can be used formany coupling reactions during which the reactivity of the phosphate,diphosphate polyphosphate, thiophosphate, phosphonate, thiophosphonate,carboxylate, sulphate, mercapto or silantriol group-containingderivatives is applicable for binding tissue-specific bindingsubstances, pharmacologically active substances, pharmacologicallyactive cells, pharmacologically active complexing agents, cell-fusioningsubstance and/or substance acting as gene-transfer medium.

The superparamagnetic particles according to the invention, which werestabilised with amino acid group-containing stabiliser substances can beused for many coupling reactions during which the reactivity of theamino acid groups is applicable for chemical binding of tissue-specificbinding substances, pharmacologically active substances,pharmacologically active cells, pharmacologically active complexingagents, cell-fusioning substance and/or substance acting asgene-transfer medium.

The superparamagnetic particles according to the invention, which werestabilised with silicate group-containing substances of ortho-silicicacid and their condensation products with organic acids and bases, canbe used for adsorptive bindings and for many coupling reactions duringwhich the reactivity of the functional groups of the organic acids andbasis which have formed stable condensation products with the silicategroups, for chemical binding of tissue-specific binding substances,pharmacologically active substances, pharmacologically active cells,pharmacologically active complexing agents, cell-fusioning substanceand/or substance acting as gene-transfer medium is applicable.

Coupling to the superparamagnetic particles of tissue-specific bindingsubstances, pharmacologically active substances, pharmacologicallyactive cells, pharmacologically active complexing agents, cell-fusioningsubstances or substances acting as gene-transfer medium furthermore hasthe advantage that by way of relaxation-time changes of theresonance-capable hydrogen atoms in the body, the therapy progress canbe observed by means of nuclear spin diagnostics.

Manufacture of superparamagnetic particles is by precipitation from asolution of ferric salt with an alkaline lye or aqueous ammonia and asubsequent targeted agglomeration of the resulting superparamagneticone-domain particles. Thereby, the superparamagnetic one-domainparticles are stirred in water and, at a pH value of 1 to 7, brought toaggregation, by heating to 80 to 120° C.--in the case of temperaturesexceeding 100° C. in an autoclave.

After cooling the dispersion, the particles are washed until such timeas the electrical conductivity of the filtrate is <10 μS/cm. Thesuperparamagnetic particles thus manufactured immediately form a rapidlysedimenting precipitation which even through vigorous stirring or ultrasound treatment cannot be transformed into a stable dispersion. Onlybinding of stabiliser substances at the surface of the superparamagneticparticles provides dispersability.

In the case of some stabiliser substances it is sufficient to stir witha glass rod, other stabiliser substances need higher energy input, suchas for example heating or the effect of ultrasound, in order to obtainstable dispersions.

Depending on the field of application, the magnetic dispersions can bedialyzed in order to remove the excess stabiliser substance.

The stabilised superparamagnetic particle dispersions contain thesuperparamagnetic one-domain particles which are not yet aggregated oronly weakly aggregated. These form a stable magnetic liquid which iseasily separated from the larger superparamagnetic particles by theirsedimentation in a magnetic field of corresponding strength andinhomogeneity. The stabilised superparamagnetic one-domain particles areeasily used as a contrast medium for nuclear spin diagnostics, as cellfusioning substances or as gene-transfer medium whereby here too, theefficacy of cell fusion and gene transfer can be examined by way ofnuclear spin diagnostics.

In a simple embodiment of magnetic separation, a glass beaker of themagnetic dispersion is placed on a permanent magnet with a magnetic fluxdensity of 0.1 T and after a sedimentation period of approx. 30 min, themagnetic liquid above the sediment is poured away. The superparamagneticaggregates remain. Depending on particle size they spontaneouslydisperse again in the dispersion or remain as sediment in the glassbeaker. Up to a particle size of approx. 500 nm the superparamagneticparticles disperse again spontaneously or through gentle stirring in theaqueous dispersion medium.

Sedimentation stability of the superparamagnetic aggregates according tothe invention is significantly higher than in the magnetic particles,known so far, with comparable magnetic properties. This can probably beattributed to the pronounced structuring of the water moleculessurrounding the superparamagnetic particles and the resulting increasedStokes' particle diameter.

Since the proportion of superparamagnetic one-domain particles in thesuperparamagnetic aggregates is significantly higher than in themagnetic particles known so far, the precipitation rate of thesuperparamagnetic aggregates in an inhomogenous magnetic field is alsohigher. In a 10% by weight aqueous dispersion of superparamagneticparticles, with a diameter of approx 100 nm and a magnetite content of95%, the precipitation time of the magnetic particles on a permanentmagnet with a magnetic flux density of 0.1 T is less than 1 min.

As aggregates, the superparamagnetic particles according to theinvention have an iron oxide content of 90 to 98% by weight. Whencompared to the state of technology whereby magnetic particles cancontain up to 60% by weight iron oxide, this means a significantimprovement in the magnetic properties. Thus, to achieve the samemagnetic interaction, the new superparamagnetic particles can beaccordingly smaller than the magnetic particles known up to now. Thespecific surface enlarges; more pharmacologically active substances ortissue-specific binding substances can be coupled to the surface. Adecrease in particle size also improves biological compatibility whichincreases the rate of degradability in the body. Also the free availabletime of the magnetic particles during magnetic drug targeting, i.e. thetime until the particles are bound by the reticulo endothelial system,increases with a decrease in particle size.

Depending on particle size and composition of the stabiliser substances,bio-availability of the superparamagnetic particles in the body rangesonly from a few minutes to several hours, i.e. the superparamagneticparticles are bound relatively quickly by the reticulo endothelialsystem.

By means of examples, the manufacture of superparamagnetic particlesaccording to the invention is described.

EXAMPLE 1

Dissolve iron (III) chloride (270 g) and iron (II) sulphate (153 g) in 1l distilled water. Set a pH value of 9.5 by adding caustic soda whilestirring. After completed precipitation set the pH value to 5.0 bystirring and adding hydrochloric acid and heat to 100° C. After coolingthe dispersion, wash the precipitation, until the electricalconductivity of the filtrate is <10 μS/cm. Stabilising of thesuperparamagnetic particles takes place by mixing an aqueous stabilisersolution or a stabiliser solution containing low-boiling polar solventswith the magnetic particles at room temperature. In this, the stabilisersolution, depending on the desired properties, can consist of purestabiliser substances or of mixtures of stabiliser substances. In orderto accelerate dispersion and stabilisation, the dispersion can bestirred or treated with ultrasound. If low-boiling organic solvents areused, these will be removed after stabilisation, by vacuum evaporationor dialysis.

EXAMPLE 2

Dissolve iron (III) chloride (270 g) and iron (II) chloride (119 g) in 1l distilled water. Set a pH value of 9.6 by adding ammoniacal gas liquorwhile stirring. After completed precipitation set the pH value to 6.0with hydrochloric acid, add 20 ml hydrogen peroxide (30%), and heat to100° C. After cooling, wash the precipitation with distilled water untilthe electrical conductivity of the filtrate is <10 μS/cm. The resultingg-Fe₂ O₃ can be stabilised.

EXAMPLE 3

Dissolve iron (III) chloride (270 g) and iron (II) sulphate (153 g) in 1l distilled water. By adding caustic soda while stirring set a pH valueof 9.5. After completed precipitation add 20 g polyphosphoric acid tothe dispersion while stirring and heat to 100° C. After cooling thedispersion, wash the precipitation, until the electrical conductivity ofthe filtrate is <10 μS/cm. Stir the solid matter into 300 ml of waterand disperse with ultrasound for 20 min at 100 W. Sediment the resultingdispersion for 30 min on a permanent magnet with a magnetic flux densityof 0.1 T and pour away the magnetic liquid above the sediment. Theliquid above the sediment predominantly contains stabilisedsuperparamagnetic one-domain particles. The sediment on the permanentmagnet contains the superparamagnetic degradable aggregates. By means ofrepeated washing with distilled water and renewed sedimentation in themagnetic field, the superparamagnetic aggregates can be obtained pureand with close particle size distribution. The average particle diameterof superparamagnetic particles is approx. 80 nm.

EXAMPLE 4

Stir the entire quantity of liquid above the sediment, of thesuperparamagnetic one-domain particles stabilised with polyphosphoricacid from example 3 into a solution of 20 g oxypolygelatine in 400 mlphysiological table salt solution. The resulting magnetic liquid can beused as an i.v. contrast medium for nuclear spin diagnostics.

EXAMPLE 5

Stir the entire quantity of the magnetite sediment stabilised withpolyphosphoric acid from example 3 into 300 ml water, set to a pH of 1.0with hydrochloric acid (10%), add a mixture of 28.4 g bismuth chloridein 20 ml concentrated hydrochloric acid and subsequently set the pH to2.5 by means of caustic soda. Into this dispersion stir a solution of 30g sodium silicate in 200 ml distilled water and disperse for 10 min withultrasound at 100 W. Separation of the non-agglomerated or only weaklyagglomerated superparamagnetic one-domain particles which form a stablemagnetic liquid is by magnetic sedimentation as described in example 3.The superparamagnetic one-domain particles and the superparamagneticaggregates are highly suitable as oral contrast medium for nuclear spindiagnostics and x-ray diagnostics.

EXAMPLE 6

For stabilisation, stir the entire quantity of the magnetite sedimentfrom example 1 into a solution of 30 g gallic acid into 400 ml distilledwater and disperse with ultrasound for 10 min at 100 W. Separation ofthe non-agglomerated or only weakly agglomerated superparamagneticone-domain particles which form a stable magnetic liquid is by magneticsedimentation as described in example 3. The superparamagneticaggregates are highly suitable for magnetic enrichment in tumours. Here,by means of magnetomechanical immune stimulation, or additionally byhyperthermia, i.e. by irradiation of electromagnetic radiation andwarming the tumour, they can destroy the tumour. The superparamagneticone-domain particles can be employed as an oral or i.v. contrast mediumfor nuclear spin diagnostics.

EXAMPLE 7

Place the entire quantity of the magnetite sediment from example 1 intoa solution of 40 g methoxy polyethylene glycol phosphate (MG 2000) and20 g rutinoside in 300 ml methanol and disperse with ultrasound for 5min at 100 W. Subsequently, add 500 ml distilled water to the dispersionand distil the methanol off. Disperse the aqueous solution once morewith ultrasound for 20 min at 100 W. Separation of the non-agglomeratedor only weakly agglomerated superparamagnetic one-domain particles whichform a stable magnetic liquid is by magnetic sedimentation as describedin example 3. The superparamagnetic aggregates can be employed forparenteral tumour damage because, as shown in tests with laboratory miceand rats, a strong activation of the immune system takes place which canlead to total remission of tumours. In addition, the superparamagneticaggregates are suitable for coupling to tissue-specific bindingsubstances, pharmacologically active substances and pharmacologicallyactive cells. The superparamagnetic one-domain particles can be employedas parenteral contrast medium for nuclear spin diagnostics to depict thevascular system and the gastro-intestinal tract or as a vector for genetransfer.

EXAMPLE 8

20 ml of the dispersion of the superparamagnetic aggregates from example7, with a magnetic saturation induction of 10 mT, are mixed with asolution of 10 mg doxorubicin in a solution of 10 ml physiological tablesalt. These superparamagnetic particles are very well suited formagnetic enrichment in tumours. The strong activation of the immunesystem by the superparamagnetic particles can lead to tumour damagewhich can be enhanced by the effect of the cytostatic substance.

EXAMPLE 9

Add the entire quantity of the g-Fe₂ O₃ sediment from example 2 to asolution of 30 g tannic acid in 500 ml water and stir for 5 min anddisperse for 10 min in an ultrasound dispergator (at 100 W). Dialyse theresulting dispersion with a 50 kD filter against distilled water, inorder to remove excessive stabiliser substance. Separation of thenon-agglomerated or only weakly agglomerated superparamagneticone-domain particles which form a stable magnetic liquid is by magneticsedimentation as described in example 3. The resulting superparamagneticaggregates, whose average particle diameter is approx. 80 nm, aresuitable for coupling to tissue-specific binding substances containingamino acid, to pharmacologically active substances and pharmacologicallyactive cells. The superparamagnetic one-domain particles can be employedas an oral or i.v. contrast medium for nuclear spin diagnostics.

EXAMPLE 10

20 ml of the superparamagnetic aggregates from example 9, with amagnetic saturation induction of 10 mT, are mixed with a solution of 10mg mitoxantrone in 20 ml of an acetate-buffered physiological table saltsolution.

These superparamagnetic particles are highly suitable for magneticenrichment in tumours. Here they can lead to tumour damage by magneticenrichment in the tumour and enhanced desorption of the cytostaticsubstance under the effect of the non-homogenous magnetic field.

EXAMPLE 11

Place the entire quantity of the magnetite sediment from example 1 intoa solution of 20 g oxypolygelatine in 400 ml distilled water, stir for 5min and wash until the electrical conductivity of the filtrate is <100μS/cm. Disperse the suspension with ultrasound for 10 min at 100 W.Separation of the non-agglomerated or only weakly agglomeratedsuperparamagnetic one-domain particles which form a stable magneticliquid is by magnetic sedimentation as described in example 2. Thesuperparamagnetic one-domain particles can be employed as parenteralcontrast medium for nuclear spin diagnostics to depict the vascularsystem.

EXAMPLE 12

Add the entire quantity of the magnetite sediment from example 2 to asolution of 40 ml of a solution of 40% phytic acid in 500 ml distilledwater, stir for 5 min and disperse for 10 min in an ultrasounddispergator (100 W) Titrate the resulting dispersion with a 30% solutionof sodium silicate to a pH value of 7.0, disperse for 10 min in anultrasound dispergator (100 W) and sediment for 30 min on a permanentmagnet with a magnetic flux density of 0.1 T and suck off the magneticliquid above the sediment. The sediment on the magnetic field containsthe superparamagnetic particles. By means of repeated washing withdistilled water and renewed sedimentation in the magnetic field, thesuperparamagnetic particles can be obtained pure and with close particlesize distribution. The superparamagnetic particles, having an averageparticle diameter of approx. 160 nm, are suitable for the coupling oftissue-specific binding substances containing amino acid, ofpharmacologically active substances and pharmacologically active cells.

The main application of the superparamagnetic particles according to theinvention is in the areas of magnetic drug targeting, contrast mediums,cell fusion and gene transfer.

Because of the very high proportion of magnetic material (90 to 98% byweight) even small magnetic particles can be concentrated very well andvery quickly in certain regions of the body, by means of electromagneticor permanent-magnetic fields. The superparamagnetic aggregates accordingto the invention can be employed for parenteral tumour damage, becauseinjection of these aggregates into the bloodstream causes strongactivation of the immune system which can lead to total remission oftumours. By coupling pharmacologically active substances to thesuperparamagnetic aggregates, their concentration at the place of actioncan be increased, in particular when using tumour-specific antibodies.This circumstance is significant for cancer therapy, because thesubstances used in chemotherapy of tumours have very severe side-effectson the entire organism and because with enrichment at the point ofaction the remaining body is not burdened as much with cytostaticsubstances or radioactive isotopes.

When coupling nucleides, nucleic acids, antimetabolites or antitumoralor antiviral derivatives to the superparamagnetic aggregates, theirconcentration at the place of action can be drastically increased, inparticular when employing them in gene therapy. By coupling them toviruses, bacteria, cells and their surface molecules, thesuperparamagnetic particles can be employed for immune-activation in thebody, whereby the effect of magnetic fields supports immune activation.

In magnetic drug targeting, the quantity of superparamagnetic particlesto be used is dependent on the particle size, the composition of thestabiliser substances, the presence of binding-specific antibodies andthe strength of the magnetic field at the place of action. Thesuperparamagnetic particles according to the invention may be used asoral and parenteral contrast medium for nuclear spin diagnostics. Whenused as parenteral contrast medium the quantity of superparamagneticparticles for the MRI is around 20 mM Fe/kg body weight and when used asan oral contrast medium for the MRI at approx. 10 mM Fe/kg body weight.

In animal experiments, good contrast effects were achieved as aparenteral contrast medium for liver, spleen, bone marrow and bloodcirculation, for lymphography, as antibody-specific contrast medium fortumour and thrombosis diagnostics and as an oral contrast medium fordepicting the gastro-intestinal tract.

The superparamagnetic particles can also be used for in-vitrodiagnostics, cell fusion and for gene transfer, optionally with theeffect of magnetic fields, if the respective diagnostics, cell-fusioningand gene-transferring substances are bound to the particle surface. Dueto the strong magnetic interaction of the superparamagnetic particleswith magnetic fields, even very small superparamagnetic particles caneasily be separated from the reaction mixture after completion of thediagnostic reaction, cell fusion or gene transfer.

The superparamagnetic particles can also be employed as magnetic ionexchangers and magnetic adsorbents for the separation of ions, organicmolecules, macromolecules, cells, viruses etc. in biotechnology,waste-water purification or other mass transfer processes, if therespective ion exchanger groups and adsorbents are bound to the surfacesof the particles.

I claim:
 1. Superparamagnetic particles, consisting of particlesselected from the group consisting of(a) small superparamagneticone-domain particles of iron hydroxide, iron oxide hydrate, iron oxide,mixed iron oxide or iron with a particle size ranging between 3 and 50nanometers; and (b) dispersion stable, physiological degradableaggregates with a particle size ranging between 10 and 1000 nanometers,whereby the aggregate consists of several small superparamagneticone-domain particles of iron hydroxide, iron oxide hydrate, iron oxide,mixed iron oxide or iron with a particle size ranging between 3 and 50nanometers; and the mixtures of (a) and (b); wherein to the surfaces ofthe particles or aggregates or mixtures thereof are bound (c) substancesof the group of(i) mono or polyhydroxylic group-containing or bothgroups containing aromatic substances, selected from among benzenoids,coumarins, lignans, terphenyls, flavonoids, tannins, xanthenes,benzophenones, naphthalenes, naphthoquinones, anthraquinones,anthracyclines, polycyclic condensated aromatic compounds and thephosphate, diphosphate, polyphosphate, thiophosphate, phosphonate,thiophosphonate, carboxylate, sulphate, mercapto or silantriolgroup-containing derivatives thereof.
 2. Superparamagnetic particlesaccording to claim 1, wherein the superparamagnetic one-domain particles(a) and the particles of the stable, degradable aggregates (b) compriseiron hydroxide, iron oxide hydrate, γ-Fe₂ O₃, Fe₃ O₄, mixed iron oxidesof the general formula mMO.nFe₂ O₃, where M represents the bivalentmetal ions Fe, Co, Ni, Mn, Be, Mg, Ca, Ba, Sr, Cu, Zn, Pt or mixturesthereof, from the mixed oxides of the general formula mFe₂ O₃.nMe₂ O₃,where Me represents the trivalent metal ions Fe, Al, Cr, Bi, rare earthmetals or mixtures of them.
 3. Superparamagnetic particles according toclaim 1, wherein bound to the superparamagnetic particles are substancesselected from the groups(i) a tissue-specific binding substance from thegroup of antigens, antibodies, ribonucleic acids, desoxyribonucleicacids, ribonucleic acid sequences, desoxyribonucleic acid sequences,hapten, avidin, streptavidin, protein A, protein G, endotoxin-bindingproteins, lectine, and selectine; (ii) a pharmacologically activesubstance from the group of antitumour proteins, enzymes, antitumourenzymes, antibiotics, plant alkaloids, alkylation reagents,antimetabolites, hormones and hormone antagonists, interleukins,interferons, growth factors, tumour necrosis factors, endotoxins,lymphotoxins, urokinase, streptokinase, plasminogen-streptokinaseactivator complex, tissue plasminogen activators, desmodus plasminogenactivators, macrophagen activating bodies, antisera, proteaseinhibitors, substances containing radioactive isotopes, tensides,cardiovascular pharmaceutic products, chemotherapeutic products,gastrointestinal pharmaceutic products, and neuropharmaceutic products;(iii) pharmacologically active cells from the group of the organelles,viruses, microbes, algae, fungi, in particular erythrocytes,thrombocytes, granulocytes, monocytes, lymphocytes, and Langerhansislands; (iv) a pharmacologically active complexing agent from the groupof the polycarbonic acids, polyaminocarboxylic acids, porphyrins, andcatecholamines; (v) cell-fusioning substances from the group ofpolyalkylene glycols, alkyl polyalkylene glycols, aryl polyalkyleneglycols, and alkyl aryl polyalkylene glycols; (vi) gene-transfer mediafrom the group of polyalkylene glycols, and poly imines; and mixtures ofsubstances of the groups (i)-(vi).
 4. Pharmacologically activepreparation, consisting of a pharmacologically acceptable carrier andsuperparamagnetic particles according to claim 1 with the particle sizeof the one-domain particles between 3 and 50 nm and the aggregatesranging from 10 to 1000 nm, coupled with a tissue-specific bindingsubstance, a pharmacologically active substance, a pharmacologicallyactive cell, a pharmacologically active complexing agent, acell-fusioning substance or a gene-transfer medium or a mixture thereofto destroy tumors, to stimulate the immune system, for magnetic drugtargeting, for cell fusion, for gene transfer, as a contrast medium, asa magnetic ion exchanger and as magnetic adsorbents for separationprocesses.
 5. Superparamagnetic particles, consisting of particlesselected from the group consisting of(a) small superparamagneticone-domain particles selected from the group consisting of ironhydroxide, iron oxide hydrate, iron oxide, mixed iron oxide, and ironwith a particle size ranging between 3 and 50 nanometers; and (b)dispersion stable, physiological degradable aggregates with a particlesize ranging between 10 and 1000 nanometers, whereby the aggregatecomprises several small superparamagnetic one-domain particles selectedfrom the group consisting of iron hydroxide, iron oxide hydrate, ironoxide, mixed iron oxide or iron with a particle size ranging between 3and 50 nanometers; and the mixtures of (a) and (b); wherein to thesurfaces of the particles or aggregates or mixtures thereof are bound onbinding sites on said surfaces, (c) substances selected from the groupconsisting of(i) mono or polyhydroxylic group-containing or both groupscontaining aromatic substances, selected from the group consisting ofbenzenoids, coumarins, lignans, terphenyls, flavonoids, tannins,xanthenes, benzophenones, naphthalenes, naphthoquinones, anthraquinones,anthracyclines, polycyclic condensated aromatic compounds and theirphosphate, diphosphate, polyphosphate, thiophosphate, phosphonate,thiophosphonate, carboxylate, sulphate, mercapto or silantriolgroup-containing derivatives; and in addition to the substances (c) andon their binding sites are chemically bound organic substances (d),selected from the group consisting of (vi) phosphate, diphosphate,carboxylate, polyphosphate, thiophosphate, phosphonate, thiophosphonate,sulphate, sulphonate, mercapto, carboxylate silantriol, trialkoxy silanegroup-containing polyalkylene glycols and carbohydrates; and (vii)phosphate group-containing nucleotides, the oligomers thereof or thepolymers thereof; and (viii) the alkyl, aryl, alkyl-aryl-polyethyleneglycol phosphates or -phosphonates, phosphates or phosphonates of thesubstance copolymerides from polyethylene glycol (PEG) and polypropyleneglycol (PPG), selected from among the substance copolymerides (PEG)_(n)-(PEG)_(m), (PEG)_(n) -(PPG)_(m) -(PEG)_(n), (PPG)_(m) -(PEG)_(n)-(PPG)_(m) ; and whereby n and m are positive whole numbers, selectedfor PEG in the range of 4 to 1000, for PPG in the range of 3 to 12 andfor PEG-PPG substance copolymerides in the range of 3 to 140; (ix)nitrogenous polysaccharides, selected from among mucopolysaccharides,glycoproteids, chitins, as well as their derivatives and denaturationproducts; and mixtures of substances of the groups (vi)-(ix).